INACTIVATION OF RSMA LEADS TO OVERPRODUCTION OF EXTRACELLULAR PECTINASES, CELLULASES, AND PROTEASES IN ERWINIA-CAROTOVORA SUBSP CAROTOVORA IN THE ABSENCE OF THE STARVATION CELL DENSITY-SENSING SIGNAL, N-(3-OXOHEXANOYL)-L-HOMOSERINE LACTONE

The soft-rotting bacterium, Erwinia carotovora subsp. carotovora 71, p roduces extracellular enzymes such as pectate lyase isozymes (Pels), c ellulase (Cel), polygalacturonase (Peh), and protease (Prt). While the extracellular levels of these enzymes are extremely low when the bact erium is grown in salts-yeast extract-glycerol (SYG) medium, the enzym atic activities are highly induced in SYG medium supplemented with cel ery extract. By transposon (mini-Tn5) mutagenesis, we isolated a RsmA( -) mutant, AC5070, which overproduces extracellular enzymes; the basal levels of Pel, Peh, and Cel in AC5070 are higher than the induced lev els in the RsmA(+) parent, AC5047. While Peh production is mostly cons titutive in AC5070, Pel, Gel, and Prt production is still inducible wi th celery extract. The high basal levels of pel-1, pel-3, and peh-1 mR NAs in AC5070 demonstrate that overproduction of the pectolytic enzyme s is due to the stimulation of transcription. Using chromosomal DNA fl anking mini-Tn5 as a probe, we cloned the wild-type rsmA(+) allele, wh ich suppresses Pel, Feb, Cel, and Prt production in both RsmA(+) and R smA(-) strains. The RsmA(-) mutant, like its parent, produces N-(3-oxo hexanoyl)-L-homoserine lactone (HSL), a starvation/cell density-sensin g signal required for extracellular enzyme production. To examine the role of HSL, we constructed HSL-deficient strains by replacing hslI, a locus required for HSL production, with hslI::Tn3HoHo1-Spc. While the basal levels of Pel, Peh, Cel, and Prt are comparable in the RsmA(-) mutant and its HSL(-) derivative, these enzymes are barely detectable in the Hsl(-) derivative of the RsmA(+) parent strain. The Hsl(-) RsmA (+) strain fails to elicit soft rot, whereas the Hsl(-) RsmA(-) strain , like its Hsl(+) RsmA(-) parent, remains hypervirulent. These finding s demonstrate that the RsmA(-) mutant produces extracellular enzymes a nd macerates plant tissue in the absence of HSL. We conclude that over production of extracellular enzymes in an HSL-independent manner occur s because of the inactivation of a global repressor locus, rsmA.